Fig 2: HepG2.2.15 cells were transfected by siRNA. (A) The framework of gene silencing with siRNA. (B) The relative mRNA levels of PCYT1A and LPP1. (C) The western blot analysis of LPP1, PCYT1A protein expression levels in the siRNA negative control group and treatment group, the initial figure is shown in Fig. S3. (D) The relative levels of HBsAg. (E) The relative levels of HBeAg. (F) The relative levels of HBV DNA. (G) The proliferation of HepG2.2.15 cells transfected by siRNA was assayed using cell-counting assay. (B,D–F) data are shown as mean ± SD, n = 3, t-test ***p value < 0.001, **p value < 0.01, *p value < 0.05. PA: phosphatidic acid, DAG: diglyceride, PC: phosphatidylcholine. siRNA: siPCYT1A or siLPP1, siNC: negative control siRNA.

Fig 3: Myeloid cell-specific deletion of Pcyt1a leads to improved systemic glucose metabolism on the Lepob/ob genetic background.(A) Schema of the BMT study design. (B) Body weight gain curves and (C) weights of indicated tissues of Lepob/ob mice transplanted with Pcyt1afl/fl (n = 7) or Pcyt1afl/fl Lyz2Cre/+ (n = 8) bone marrow. (D)GTT curves and areas under curve (AUC), normalised to basal glucose levels. (E) ITT curves, presented as percentage values of basal glucose levels, and areas above curve (AAC), normalised to basal glucose levels.

Fig 4: Immunostaining of Endogenous PCYT1A in Selected Mouse Tissues(A) Simplified schematic of the two major PC biosynthetic pathways in eukaryotes. The CDP-choline (Kennedy) pathway is shown in red and the methylation pathway in blue. Key enzymes discussed in the text are shown in upper case (mammals) or lower case (yeast). Pct1/CCTa/PCYT1A, choline-phosphate cytidylyltransferase A; P-Cho, phosphocholine; CDP-Cho, cytidine diphosphate-choline; CDP-Etn, cytidine diphosphate-ethanolamine; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Cho, choline.(B) (i) Immunostaining of adult mouse retina indicates that PCYT1A localizes to the nuclear membrane in the outer nuclear layer. SC, sclera; CH, choroid; RPE, retinal pigmented epithelium; OS, outer segments of rods and cones; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (ii) Zoomed-in images of the indicated field in (i).(C) (i) In the femoral growth plate of 15-day-old mice, PCYT1A localizes to the nuclear membrane of chondrocytes in the hypertrophic (HZ) but not in the resting (RZ) or proliferative (PZ) zone of growth plates. (ii) Zoomed in images of the indicated field in (i).(D) In ad-libitum chow-fed adult mice, PCYT1A localizes to the nuclear membrane in wild-type (WT) but not in Mtp knockout hepatocytes, which have impaired lipoprotein synthesis.(E) (i) PCYT1A localizes to the intranuclear region of adult mouse inguinal white adipocytes but translocates to the nuclear membrane upon adipogenic induction in OP9 cells (ii). Lipid droplets (LDs) were stained with BODIPY (green) as described in the STAR Methods. D0–D3 indicate day after onset of differentiation.Scale bars, 20 µm. See Figure S1.

Fig 5: The relative mRNA expression levels of major genes in PC synthesis and metabolic pathways. (A) Phosphatidylcholine synthesis and metabolism pathways. (B) The relative mRNA level of major genes in phosphatidylcholine synthesis and metabolism in HepG2 and HepG2.2.15 cells, n = 3, detailed data are shown in Table S4. (C) The western blot analysis of LPP1, PCYT1A protein expression levels in HepG2 and HepG2.2.15 cells, the initial figure is shown in Fig. S2. (D) The levels of the substrates of the PC synthesis in HepG2 and HepG2.2.15 cells, n = 10. (B,D) data are shown as mean ± SD, t-test, ***p value < 0.001, **p value < 0.01, *p value < 0.05. PA: phosphatidic acid, DAG: diglyceride, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine, LPC: lyso-phosphatidylcholine, GPC: glycerol-phosphorylcholine.